Levodione reductase

Abstract

 A levodione reductase having the following physico-chemical properties is provided: molecular weight: 142,000 - 155,000 ± 10.000 (consisting of four homologous subunits having a molecular weight of 36,000 ± 5,000); co-factor: nicotinamide adenine dinucleotide (NAD/NADH); substrate specificity: active on levodione; optimum temperature: 15 - 20°C at pH 7.0; optimum pH: 7.5; and activator: K⁺, Cs⁺, Rb⁺, Na⁺ and NH₄⁺. Said levodione reductase, which is capable of producing actinol, an important intermediate for the production of zeaxanthin, from levodione, can be produced from a microorganism belonging to the genus Corynebacterium, preferably the microorganism Corynebacterium aquaticum AKU 611 (FERM BP-6448) or a functional equivalent, subculture, mutant or variant thereof.

Description

[0001] The present invention relates to a novel enzyme, namely levodione reductase (hereinafter referred to as LR), a process for producing the enzyme and a process for producing (4R, 6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (hereinafter referred to as actinol) from (6R)-2,2,6-trimethylcyclohexane-1,4-dione (hereinafter referred to as levodione) utilizing the enzyme. Actinol is an important intermediate for the production of zeaxanthin.

[0002] In European Patent Application No. 98115564.1, filed on August 19, 1998, and in the later European application, No. 99115723.1, filed on August 16, 1999 and claiming the priority of the earlier one, there is disclosed a process for the manufacture of actinol which comprises contacting levodione with a microorganism which is selected from the group consisting of microorganisms of the genera Cellulomonas, Corynebacterium, Planococcus and Arthrobacter and which is capable of the selective asymmetric reduction of levodione to actinol, and recovering the resulting actinol from the reaction mixture. Corynebacterium aquaticum AKU611 (FERM BP-6448) was found to be one of the best microorganism strains for this purpose.

[0003] The microorganism strain Corynebacterium aquaticum AKU611 has the following taxonomical properties:

1) Growable temperature :	15-40°C
2) Optimum temperature for growth :	30°C
3) Obligatory aerobic and gram negative microorganism
4) Spore formation :	None
5) Polymorphism and traditional rod-cocus cycles can be observed during cultivation.
6) Motility :	None

[0004] Moreover, the strain Corynebacterium aquaticum AKU611 was identified as such based on assimilation of various carbon sources by the Biolog System (Biolog Inc., 3447 Investment Blvd., Suite 3, Hayward, California 94545, USA : Nature Vol. 339, 157-158, May 11, 1989) as follows: Cells of the strain were inoculated with 96-well microtiter-plates and incubated for 24 hours at 28°C. Each well contained one of 96 kinds of carbon sources in BUGM+B medium (Biolog Universal Growth Media + Blood; Biolog Inc.).

[0005] After incubation, the strain showed the following assimilation of carbon sources:

[0006] It is an object of the present invention to provide the novel LR which acts on levodione to produce actinol. LR has the following physico-chemical properties:

a) Molecular weight: 142,000 - 155,000 ± 10,000
(consisting of four homologous subunits having a molecular weight of 36,000 ± 5,000)

b) Co-factor: nicotinamide adenine dinucleotide (NAD/NADH)

c) Substrate specificity: active on levodione

d) Optimum temperature: 15 - 20 °C at pH 7.0

e) Optimum pH: 7.5

f) Activator: K⁺, Cs⁺, Rb⁺, Na⁺ and NH₄⁺

[0007] It is another object of the present invention to provide a process for producing the novel LR as defined above by cultivation of a microorganism belonging to the genus Corynebacterium, which is capable of producing the LR having the above physico-chemical properties, in an aqueous nutrient medium under aerobic conditions, disrupting the cells of the microorganism and isolating and purifying the LR from the cell-free extract of the disrupted cells of the microorganism. A still further object of the present invention is to provide a process for producing actinol from levodione utilizing the LR, which comprises contacting levodione with (i) an LR as defined above in the presence of the reduced form of nicotinamide adenine dinucleotide (NADH), or (ii) a cell-free extract of said microorganism, and in each of the cases (i) and (ii) isolating the resulting actinol from the reaction mixture.

[0008] The physico-chemical properties of the purified sample of the LR prepared according to the Examples presented below are as follows:

1) Enzyme activity

[0009] The novel LR of the present invention catalyzes the reduction of levodione to actinol in the presence of a co-factor according to the following formula:

[0010] The reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) does not work as an electron donor in this reaction system.

[0011] The standard enzyme assay was performed as follows: The basal reaction mixture of total volume 500 µl and consisting of 100 µl of 1 M potassium phosphate buffer (pH 7.0), 20 µl of 8 mM NADH in 0.2 mM KOH, 10 - 40 µl of the enzyme solution, and water up to a total of 500 µl, was incubated for 1 minute at 37 °C. Then 2 µl of 0.5 M levodione solution were added to give a final concentration of 2 mM, and the whole was incubated for 1 minute at 37 °C. The enzyme activity was monitored with the decrease of the absorbance of NADH at 340 nm. One unit of the enzyme activity was defined as the amount of the enzyme which catalyzes the oxidation of 1 µmole of NADH per minute.

[0012] NAD, NADH and NADPH are available from Oriental Yeast, 3-6-10 Azusawa, Itabashi-ku, Tokyo, Japan.

[0013] The protein concentration was determined by using a Bio-Rad protein assay kit (Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547, USA)

2) Molecular weight

[0014] The molecular weight (MW) of the enzyme was measured with a gel filtration HPLC column Cosmosil 5Diol-300 (nacalai tesque: Nishi-iru, Karasuma, Nijodohri, Nakagyouku, Kyoto-fu, Japan). The apparent molecular weight of the (whole) enzyme was calculated to be 142,000 - 155,000 ± 10,000 in comparison with the molecular weight marker proteins: LMW + HMW gel filtration calibration kit, Amersham Pharmacia Biotech (SE-75184 Uppsala, Sweden); ferritin (MW 440,000), aldolase (MW 158,000), bovine serum albumin (MW 67,000), ovalbumin (MW 43,000), and ribonuclease A (MW 13,700). SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) gave a single band with a molecular weight of 36,000 ± 5,000 in comparison with the molecular weight marker proteins: LMW Electrophoresis calibration kit, Amersham Pharmacia Biotech; bovine serum albumin (MW 67,000), ovalbumin (MW 43,000), carbonic anhydrase (MW 30,000), soybean trypsin inhibitor (MW 20,100), and α-lactalbumin (MW 14,400). This indicates that the enzyme is composed of four homologous subunits. The values of the molecular weight of the whole enzyme (142,000-155,000 ± 10,000) and of each subunit (36,000 ± 5,000) were determined as accurately as the respective methods, i.e. the gel filtration column method and the SDS-PAGE method, allowed.

3) Co-factor

[0015] The co-factor requirement of the enzyme to convert levodione to actinol was investigated. As a result, it was established that NADH could serve as a co-factor for this reductive reaction, but that NADPH could not.

4) Substrate specificity

[0016] The substrate specificity of the enzyme was determined using the same enzyme assay method as described under 1), except that various substrate solutions (2mM, final concentration in the reaction mixture) were used instead of levodione. It was shown that levodione was the only substrate for which the enzyme exhibited activity.

Table 1

Substrate	Enzyme Activity (unit/ml)
Levodione	5.66
Cyclohexanone	Not detected (ND)
1,2-Cyclohcxane-dione	ND
1,3-Cyclohexane-dione	ND
1,4-cyclohexane-dione	ND
Cyclopentanone	ND
2-Cyclohexene	ND
1,5,5-Trimethylcyclohexene	ND
1,3-Cyclopentane-dione	ND
DL-Mevalonolactone	ND
D-Camphor	ND
L-Camphor	ND
Maleic anhydride	ND
4-Chloro-3-oxo-butanoic acid ethyl ester	ND

5) Optimum temperature

[0017] The enzyme activities were measured at temperatures from 2 to 45 °C. The optimum temperature of the enzyme activity was 15 - 20 °C

Table 2

Temperature (°C)	Relative Activity (%)
2	77.8
5	83.8
10	89.1
15	100
20	92.4
25	90.9
30	78
35	70.7
40	59.4
45	31.6

6) Optimum pH

[0018] The correlation between the enzyme activity and the pH values of the reaction mixture was determined by using the same enzyme assay method as described under 1), except that various pHs and buffers were used and 40 µl of 2.5M KCl solution were added to the reaction mixture. The optimum pH of the enzyme reaction was found to be 7.5.

Table 3

Buffer	pH	Relative Activity (%)
Potassium phosphate buffer	5.5	59.3
	6.0	68.5
	7.0	78.4
	7.5	100
[4-(2-Hydroxyethyl)piperazino]-ethanesulphonic acid (HEPES)	7.0	7.2
	7.5	10.0
	8.0	21.7
	8.5	21.9
Tris-HCl	8.5	6.5
	9.0	3.9
	10.0	1.3

7) Effect of metal ions

[0019] The effect of metal ions on the enzyme activity was investigated by using the same enzyme assay method as described under 1), except that 100 µl of 1 M Tris-HCl buffer (pH 7.5) were used instead of 100 µl of 1 M potassium phosphate buffer (pH 7.0), and various metal solutions were added to the reaction mixture to give a final concentration of metal between 100 mM and 3 M. As a result, it was established that the enzyme activity was increased about 250-fold in the presence of 3 M RbCl and 1.8 M CsCl.

Table 4-1

Metal	Concentration (mM)	Relative Activity (%)
NaCl	3	0.4
RbCl (+ 3mM NaCl)	100	6.4
	200	7.5
	400	23.3
	800	32.6
	1600	66.4
	1800	65.1
	2000	93.2
	2500	93.3
	3000	100
CsCl(+ 3mM NaCl)	100	5.9
	200	14.3
	400	28.3
	800	48.1
	1600	71.4
	1800	100
	2000	86.8
	2500	88.4
	3000	88.0

Table 4-2

Metal	Concentration (mM)	Relative Activity (%)
None	0	2.8
KCl	200	11.5
	400	22.2
	800	40.5
	1600	45.3
	1800	33.5
	2000	28.4
	2500	25.9
	3000	25.2
NH4Cl	100	6.4
	200	21.3
	400	39.3
	800	67.1
	1600	100
	1800	75.3
NaCl	100	4.2
	200	7.7
	400	19.7
	800	35.9
	1600	75.3
	1800	62.8
	2000	62.8
	2500	53.9
	3000	34.1

8) Temperature stability

[0020] The enzyme solution was treated at various temperatures for 10 minutes, and the remaining enzyme activities were measured by using the same enzyme assay method as described under 1). It was established that the enzyme was stable up to 35°C, and deactivated with increasing temperature, becoming completely de-activated at 55 °C.

Table 5

Temperature (°C)	Relative Activity (%)
0	100
30	93.3
35	85.1
40	47.8
45	28.9
50	3.2
55	0

9) pH stability

[0021] The enzyme was treated in 1 M buffers of various pHs for 10 minutes at 30°C, and its remaining activity was measured by using the same enzyme assay method as described under 1). The enzyme was found to be most stable in the pH range between 8.0 and 8.5.

Table 6

Buffer	pH	Relative Activity (%)
Bis-tris	6	40.6
	7	71.9
HEPES	7	63.4
	7.5	61.6
	8	91.6
Tris-HCl	8.5	100
	9	85.3

10) Michaelis constant (Km) and Maximum velocity (Vmax) values

[0022] The Km and Vmax values of the enzyme were measured by using levodione and actinol as the substrates. The basic enzyme assay method is the same as described under 1), but the substrate and the enzyme concentrations were varied. The Km and Vmax values against levodione as the substrate were 8.5 mM and 101.26 unit/mg, respectively. On the other hand, the Km and Vmax values against actinol as the substrate were 1.36 mM and 15.91 unit/mg, respectively.

[0023] The Km and Vmax values were calculated on the basis of the known Michaelis-Menten equation. Km is the concentration of the substrate that gives 50% of the Vmax of the enzyme reaction. The values give a useful indication of the catalytic properties of the enzyme for the involved substrate.

11) Purification procedure

[0024] The purification of the LR may in principle be effected by any combination of known purification methods, such as fractionation with precipitants, e.g. ammonium sulfate, polyethylene glycol and the like, ion exchange chromatography, adsorption chromatography, gel-filtration chromatography, gel electrophoresis and salting out and dialysis.

[0025] As mentioned above, the LR provided by the present invention can be prepared by cultivating an appropriate microorganism in an aqueous nutrient medium under aerobic conditions, disrupting the cells of the microorganism and isolating and purifying the LR from the cell-free-extract of the disrupted cells of the microorganism.

[0026] The microorganisms used for the present invention are microorganisms belonging to the genus Corynebacterium which are capable of producing LR as defined hereinbefore. Functional equivalents, subcultures, mutants and variants of said microorganism can also be used in the present invention.

[0027] A preferred strain is Corynebacterium aquaticum. The specific strain most preferably used in the present invention is Corynebacterium aquaticum AKU611 (FERM BP-6448), a sample of which was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan, on August 4, 1998, under the Budapest Treaty.

[0028] Furthermore, European Patent Application No. 98115564.1, filed on August 19, 1998, and the later European application, No. 99115723.1, filed on August 16, 1999 and claiming the priority of the earlier one, disclose some characteristics of this strain.

[0029] The microorganism may be cultured in a nutrient medium containing saccharides such as glucose and sucrose, alcohols such as ethanol and glycerol, fatty acids such as oleic acid and stearic acid, or esters thereof, or oils such as rapeseed oil and soybean oil as carbon sources; ammonium sulfate, sodium nitrate, peptone, amino acids, corn steep liquor, bran, yeast extract and the like as nitrogen sources; magnesium sulfate, sodium chloride, calcium carbonate, potassium monohydrogen phosphate, potassium dihydrogen phosphate and the like as inorganic salt sources; and malt extract, meat extract and the like as other nutrient sources. The cultivation can be carried out aerobically, normally for a period of 1 to 7 days at a medium pH of 3 to 9 and a cultivation temperature of 10 to 40°C.

[0030] An embodiment for isolation and purification of the LR from the microorganism after the cultivation is as follows:

(1) Cells are harvested from the liquid culture broth by centrifugation or filtration.

(2) The harvested cells are washed with water, physiological saline or a buffer solution having an appropriate pH.

(3) The washed cells are suspended in the buffer solution and disrupted by means of a homogenizer, sonicator, French press or treatment with lysozyme and the like to give a solution of disrupted cells.

(4) The LR is isolated and purified from the cell-free extract of disrupted cells.

[0031] The LR provided by the present invention is useful as a catalyst for the production of actinol from levodione.

[0032] The reaction of the LR-catalysed reduction of levodione to actinol is conveniently conducted at pH values of from about 6.0 to about 9.0 in the presence of NADH in a solvent. As a solvent, any buffer which maintains the pH in the range of about 6.0 to about 9.0, such as Tris-HCl buffer, phosphate buffer, Bis-tris buffer, HEPES buffer and the like, is suitable.

[0033] A preferred temperature range for carrying out the reaction is from about 2 to about 30 °C. The reaction usually gives the best results when the pH and the temperature are in the ranges about 7.0 to 8.0 and 10 to 25 °C, respectively.

[0034] The concentration of levodione in the solvent depends on the other reaction conditions, but in general is from 1 mM to 2 M, preferably from 10 mM to 100 mM.

[0035] The amount of the LR and NADH suitably present in the reaction mixture depends on the other reaction conditions, but in general is in each case independently about 10^-4 to 10^-6 of the amount of the substrate. When a regeneration system of NADH from NAD is coupled with the above reaction system, the reaction proceeds more efficiently.

[0036] In the reaction, the LR may also be used in an immobilized state with an appropriate carrier. Any means of immobilizing enzymes generally known in the art may be used. For instance, the enzyme may be bound directly to a membrane, granules or the like of a resin having one or more functional groups, or it may be bound to the resin through bridging compounds having one or more functional groups, e.g. glutaraldehyde. Such enzyme immobilizing means are described for example on pages 369-394 of the 2^nd Edition of Microbial Enzymes and Biotechnology (Elsevier Applied Science 1990; Ed. W.M. Fogarty and C.T. Kelly).

[0037] The following Examples further illustrate the present invention.

Example 1

Preparation of LR

[0038] All the operations were performed at 4 °C, and the buffer was 10 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM dithiothreitol unless otherwise stated.

Cultivation of Corynebacterium aquaticum AKU 611 (FERM BP-6448)

[0039] One colony of Corynebacterium aquaticum AKU 611 (FERM BP-6448) on an agar plate was inoculated into 5ml of the medium (pH 7.0) consisting of D-glucose (1%), KH₂PO₄ (0.3%), MgSO₄·7H₂O (0.02%), Peptone (1.5 %), NaCl (0.2%) and yeast extract (0.1%) in a tube, and incubated for 20 hours at 30°C. This culture was inoculated into 500 ml of the same medium as above in a 2 l Sakaguchi flask, and incubated for 20 hours at 30°C. A 250 ml portion of the seed culture was inoculated into 20 l of the same medium in a jar fermenter MSJ-U3W (Marubishi Bioengineering, 2-20-15 Higashikanda, Chiyoda-ku, Tokyo, Japan). Cultivation was effected at 30°C for 20 hours with aeration at the rate of 20 l/min. and agitation at 300 rpm. The culture thus obtained was centrifuged at 8,000 rpm for 20 minutes at 4°C. In total, 133.8 g of wet cells were obtained.

(2) Preparation of the cell-free extract

[0040] The wet cells (30 g) were suspended in 90 ml of the buffer, and sonicated for 1 hour at 190 W using a Kubota Insonator 201 sonicator (Kubota, 3-29-9 Hongo, Bunkyo-ku, Tokyo, Japan). After sonication, the sample was centrifuged at 16,000 rpm for 20 minutes. As a result, 80 ml of the cell-free extract containing 2,444 mg of protein were obtained.

(3) Ammonium sulfate precipitation

[0041] To the cell-free extract (80 ml) obtained in the previous step was added ammonium sulfate until a 60% saturation concentration had been achieved. Then the resulting precipitate was collected by centrifugation, solubilized in 15 ml of the buffer, and dialyzed four times against 5 l of the buffer. The total enzyme activity in this solution was 38.8 units.

(4) Diethylaminoethyl (hereinafter referred to as DEAE)-Sephacel column chromatography

[0042] The dialyzed sample prepared as described above was applied to a DEAE-Sephacel column (2.8 cm in diameter and 18 cm in height; Amersham Pharmacia Biotech) which was equilibrated with the buffer. After washing the column with the same buffer, the enzyme was eluted with 600 ml of a linear gradient of NaCl (0 - 0.8 M). The active fractions were collected and concentrated by ultrafiltration (ultrafilter YM-10 with Amicon concentration apparatus (Amicon Inc., Beverly, MA 01915, USA) to 10 ml.

(5) Alkyl Superose column chromatography

[0043] To the sample from the previous step was added (NH₄)₂SO₄ to a final concentration of 2 M, and the mixture was filtered. An alkyl supersose 10/10 column (1 cm in diameter and 10 cm in height; Amersham Pharmacia Biotech) was equilibrated with the buffer containing 2 M (NH₄)₂SO₄, and applied by the above sample. The enzyme was eluted by a 150 ml of linear gradient of the buffer [2 - 0 M (NH₄)₂SO₄]. The active fractions were collected, and dialyzed four times against 5 l of the buffer.

(6) MONO Q HR5/5 column chromatography

[0044] The dialyzed sample from the previous step was applied to a MONO Q 5/5 column (5 mm in diameter and 5 cm in height; Amersham Pharmacia Biotech) which was equilibrated by the buffer. The enzyme was eluted with 21 ml of a linear gradient of NaCl (0 - 0.8 M). The specific activity of the enzyme was not increased due to the de-activation of the enzyme during the dialyzation step before this chromatography. But the enzyme gave a homogenous band on SDS-PAGE analysis.

[0045] A summary of the purification steps of the enzyme is shown in Table 7.

Table 7

Step	Total Activity (unit)	Total Protein (mg)	Specific Activity (unit/mg)	Yield (%)
Cell-free extract	-	2444	-	-
Precipitate by 60% (NH4)2SO4	38.8	427	0.91	100
DEAE Sephacel	22	30	0.73	57
Alkyl Superose	15.2	0.92	16.5	39
MONO Q 5/5	5.4	0.38	14.2	14

(7) Identification of the reaction product

[0046] The reaction mixture (3.33 ml) containing 50 mg of NADH, 833 µl of 1 M potassium phosphate buffer (pH 7.0), 2 ml of the enzyme sample from the purification step of DEAE Sephacel column chromatography, and 550 µl of distilled water was incubated at 30°C. To this reaction mixture, 10 µl of 0.5 M levodione solution were added five times at 6 minute intervals. The reaction mixture was incubated for a further 5 minutes, and extracted with 1 ml of ethyl acetate. The extract was analyzed by gas chromatography [column: HR-20M (Shinwa, 50 Keisho-Machi, Fushimi-ku, Kyoto-shi, Kyoto, Japan) 0.25 mm ⌀ x 30m, column temperature: 160°C (constant), injector temperature: 250°C, carrier gas: He (about 1ml/min.)]. As a result, the product was identified as being actinol in comparison with a standard sample of actinol. When NADH was replaced with NADPH, only a trace amount of actinol was detected.

Claims

1. A levodione reductase having the following physico-chemical properties:

a) Molecular weight: 142,000 - 155,000 ± 10,000
(consisting of four homologous subunits having a molecular weight of 36,000 ± 5,000)

b) Co-factor: nicotinamide adenine dinucleotide (NAD/NADH)

c) Substrate specificity: active on levodione

d) Optimum temperature: 15 - 20 °C at pH 7.0

e) Optimum pH: 7.5

f) Activator: K⁺, Cs⁺, Rb⁺, Na⁺ and NH₄⁺

2. The levodione reductase according to claim 1, which is derived from a microorganism belonging to the genus Corynebacterium which is capable of producing the levodione reductase having the physico-chemical properties as given in claim 1.

3. The levodione reductase according to claim 2, wherein the microorganism is Corynebacterium aquaticum AKU 611 (FERM BP-6448), a functional equivalent, subculture, mutant or variant thereof.

4. A process for producing a levodione reductase having the following physico-chemical properties:

a) Molecular weight: 142,000 - 155,000 ± 10,000
(consisting of four homologous subunits having a molecular weight of 36,000 ± 5,000)

b) Co-factor: nicotinamide adenine dinucleotide (NAD/NADH)

c) Substrate specificity: active on levodione

d) Optimum temperature: 15 - 20 °C at pH 7.0

e) Optimum pH: 7.5

f) Activator: K⁺, Cs⁺, Rb⁺, Na⁺ and NH₄⁺

which comprises cultivating a microorganism belonging to the genus Corynebacterium, which is capable of producing a levodione reductase having the above physico-chemical properties, in an aqueous nutrient medium under aerobic conditions, disrupting the cells of the microorganism and isolating and purifying the levodione reductase from the cell-free extract of the disrupted cells of the microorganism.

5. The process according to claim 4, wherein the microorganism is Corynebacterium aquaticum AKU 611 (FERM BP-6448), a functional equivalent, subculture, mutant or variant thereof.

6. A process for producing actinol from levodione which comprises contacting levodione with

(i) a levodione reductase having the following physico-chemical properties:

a) Molecular weight: 142,000 - 155,000 ± 10,000
(Consisting of four homologous subunits having a molecular weight of 36,000 ± 5,000)

b) Co-factor: nicotinamide adenine dinucleotide (NAD/NADH)

c) Substrate specificity: active on levodione

d) Optimum temperature: 15 - 20 °C at pH 7.0

e) Optimum pH: 7.5

f) Activator: K⁺, Cs⁺, Rb⁺, Na⁺ and NH₄⁺

in the presence of the reduced form of nicotinamide adenine dinucleotide.
or

(ii) a cell-free extract of said microorganism,

and in each case isolating the resulting actinol from the reaction mixture.

7. The process according to claim 6, wherein the microorganism is Corynebacterium aquaticum AKU 611 (FERM BP-6448), a functional equivalent, subculture, mutant or variant thereof.